Analyses of T-cell differentiation from hemopoietic stem cells in the G0 phase by an in vitro method.

Using differential radiation sensitivity of components of mouse embryonal thymus, an in vitro method for studying T-cell differentiation from hemopoietic stem cells (HSCs) in the G0 phase was established. Intrathymic T-cell precursors present in embryonal thymus were found to be quite radioresistant (up to 20 Gy), and consequently 25-Gy-irradiated embryonal thymic lobes were used. Thymic lobes (25-Gy irradiated) taken from mouse fetuses (gestation day 15) were placed in Millipore-HA culture plates supported on squares of gelatin foam sponge in 24-well culture plates in which neonatal thymus stromal cells were cultured. HSCs (10(5) cells per well) in the G0 phase were added to these thymic lobes and cocultured at 37 degrees C in a 5% CO2/95% air incubator. Half the culture medium was changed every week. After 3 weeks, a large number of colonies had formed. Immunohistochemical studies and fluorescence-activated cell sorter analyses revealed that the colonizing cells regularly develop and exhibit surface markers characteristic of T cells (Thy-1, IL-2R, L3T4, Lyt-2, etc.). In situ hybridization analyses revealed that mRNA expression for T-cell receptor (TCR) beta chains occurred within colonizing cells. Using a monoclonal antibody (F23.1), expression of TCR beta-chain variable domain (V beta 8) on the surface of these developing T cells was demonstrated. These cells responded to interleukin 2 and/or anti-CD3 monoclonal antibody, indicating functional T cells. This method will be useful in studying T-cell differentiation pathways from pluripotent HSCs and in clarifying the mechanisms involved in negative and positive selection of T cells within the thymus.     

Genome macrorestriction analysis of sequential Pseudomonas aeruginosa isolates from bronchiectasis patients without cystic fibrosis.

The respiratory tracts of bronchiectasis patients may be persistently colonized with Pseudomonas aeruginosa, despite intensive chemotherapy. The organism may undergo phenotypic changes in these patients, providing misleading typing results by conventional methods. We prospectively studied eight bronchiectasis patients without cystic fibrosis over a period of 1 year. A high microbial load of P. aeruginosa was found in 70% of sputum samples collected. Of these, 55 sequential P. aeruginosa isolates were characterized by a genotyping method, pulsed-field gel electrophoresis, to overcome the problem of differentiating the P. aeruginosa strains during chemotherapy. Genome macrorestriction fingerprinting patterns were analyzed after digestion with XbaI restriction endonuclease. Of the eight patients, six harbored a single dominant strain of P. aeruginosa, with an intrapatient macrorestriction similarity pattern range of 96 to 100%. The other two patients were infected with mixed bacterial isolates including P. aeruginosa. However, diversity was observed in the P. aeruginosa isolates from all eight patients, with a relatedness of only 55 to 65%. The study further strengthens the fact that pulsed-field gel electrophoresis can be used efficiently and effectively to differentiate P. aeruginosa strains in bronchiectasis patients without cystic fibrosis.     

Monochloramine enhances Fas (APO-1/CD95)-induced apoptosis in Jurkat T cells

Monochloramine derivatives are physiological oxidants produced by activated neutrophils. We report the effects of chemically prepared monochloramine (NH2Cl) on Fas-induced apoptosis in Jurkat T cells. When the cells were pretreated with NH2Cl (20-70 microM), subsequent addition of apoptosis-inducing anti-Fas antibody resulted in a synergistic enhancement of apoptosis. Treatment of NH2Cl (50-70 microM) alone resulted in a slight but definite apoptosis. Caspase activities, as measured by DEVD and IETD cleavage activities, were also elevated synergistically by NH2Cl + anti-Fas antibody stimulation. Moreover, a broad caspase inhibitor, Z-VAD-fmk, almost completely inhibited the apoptosis induced by NH2Cl and/or anti-Fas antibody. Fas expression on the Jurkat cell surface was not affected by the NH2Cl treatment. After 3 h of NH2Cl treatment, when the apoptosis was beginning to increase, the cells showed cytochrome c release from mitochondria, proteolytic activation of caspase 9, and poly (ADP-ribose) polymerase cleavage, regardless of Fas stimulation. Z-VAD-fmk almost completely inhibited this poly (ADP-ribose) polymerase cleavage, but not cytochrome c release. By contrast, Fas stimulation alone resulted in neither cytochrome c release nor caspase 9 activation at 3 h, and the increase in the DEVD cleavage activity and apoptosis became evident at later time points. These results suggested that NH2Cl enhanced Fas-induced apoptosis through the cytochrome c release and caspase 9 activation at the early stage of apoptosis. Chloramines derived from acute inflammation may modify immune reactions, such as cell-mediated cytotoxicity and some autoimmune diseases, by the enhancement of Fas-induced apoptosis.     

Apoptosis of colorectal adenocarcinoma (COLO 201) by tumour necrosis factor-alpha (TNF-α) and/or interferon-gamma (IFN-γ), resulting from down-modulation of Bcl-2 expression

Fas antigen is constitutively expressed in the normal colon epithelium, but considerably diminished in most colorectal carcinomas. In the present study, we examine the relationship between Fas antigen expression and apoptosis using the colorectal carcinoma cell line COLO 201, on which a low grade of Fas antigen is expressed. Anti-Fas antibody had no effect on the induction of apoptosis of COLO 201. However, TNF-α and/or IFN-γ, independently and additively, up-regulated Fas antigen expression on COLO 201 and induced apoptosis in a dose-dependent manner. Both cytokines also increased the COLO 201 sensitivity to anti-Fas antibody, resulting from the down-modulation of Bcl-2 and the up-regulation of Bax. These findings indicate that cytokine(s) plus anti-Fas antibody (which mimics natural Fas ligand) are more effective in inducing apoptosis of COLO 201 than cytokine(s) alone. These findings suggest that immunotherapy in combination with cytokine(s) and lymphokine-activated killer (LAK) cells will become a more effective therapy for cancer than cytokine(s) or LAK cells alone, since the Fas ligand is expressed on activated T cells, natural killer cells and macrophages.     

何首乌组织培养的研究

采用单因子、正交设计法考察了基本培养基、激素以及光暗条件对何首乌幼嫩茎、叶诱导愈伤组织的影响,以及培养时间与愈伤组织生长量间的关系。实验研究表明:MS 2,4D1mg/L 6-BA1mg/L IBA0.5mg/L和暗培养对诱导何首乌愈伤组织产生效果较好;接种12天时愈伤组织生长量达到最大;愈伤组织经诱导能够产生完整植株,成苗率为95％。     

Aggressive Angiomyxoma of Inguinoscrotal Region Mimicking Inguinal Hernia: a Case Report

Aggressive angiomyxoma (AAM) is a rare mesenchymal tumour of pelvis and perineum, almost exclusively occurring in females. We are reporting a case of a 53-year-old gentleman who presented with a long standing inguinoscrotal swelling misdiagnosed as inguinal hernia, for which he underwent incomplete excision at local hospital and then was referred to our centre for completion surgery.     

Efficacy of oral single dose therapy with artemisinin-naphthoquine phosphate in uncomplicated falciparum malaria

All artemisinin-based combination therapies (ACTs), recommended by the World Health Organization, are 3-day regimens. A considerable level of non-compliance on ACTs has been reported from some countries. The study aimed to assess the therapeutic efficacy of single dose treatment with new generation ACT containing artemisinin plus naphthoquine. An oral single dose of eight tablets (400 mg of naphthoquine + 1000 mg artemisinin) of the combination drug was administered to adult uncomplicated falciparum malaria patients. Observations of fever, parasite clearance and reappearance, and other clinical manifestations were made on Days 0, 1, 2, 3, 7, 14, 21 and 28. Fifty-three adult falciparum positive cases, with fever or history of fever within the previous 24 h, were included in the final evaluation of the study. Mean fever clearance time, parasite clearance time were 18.2 ± 8.6 h and 34.6 ± 14.3 h, respectively. Adequate clinical and parasitological response was achieved in 52 cases, the rate being 98.1% (95% CI, 91.1-99.9). One patient was classified as late parasitological failure because of the reappearance of falciparum parasite on Day 14. The drug was well tolerated and no adverse reactions were detected in the patients. Since it is a single dose therapy, health workers can administer the drug as directly observed treatment.